The multi-drug resistance proteins (MDRs) and the multi-drug resistance associated proteins, MRP1 and MRP2 are members of the ABC-transporter superfamily, and may cause multiple drug resistance in malignant tumors. By now, at least six MRP-like proteins have been identified. The physiological role of these proteins may range from elimination of toxic agents to diverse secretory function, predominantly in epithelial cells. MRP1 is widely expressed in various tissues, while MRP2 is predominantly expressed in hepatocytes and epithelial cells of renal proximal tubules. Identifying novel compounds effectively inhibiting such transport proteins is, therefore, a major interest in trying to elaborate more effective chemotherapeutical treatment methods for drug resistant type of malignant disorders. Identifying activators of said transport proteins might also be of interest in other applications where the enhancement of their activity may help in the elimination of some undesired physiological states.
It has been previously shown that MRP1 extrudes negatively charged fluorescent compounds, e.g. calcein free acid, from the cells [Feller et al, FEBS Lett. 368, 385–388 (1995); Holló et al, FEBS Lett. 383, 99–104 (1996)]. We have recently found that this dye is rapidly expelled from cells also by MRP2. It is also possible that other members of this protein family transport this compound. In polarized cells, the distribution of these proteins is not homogeneous. Expression of MRP1 is restricted to the basolateral surface, while MRP2 is expressed solely in the apical membrane.
This feature makes possible the assessment of the function of these and other similar proteins in the so called vectorial transport assay performed on confluent cultures of polarized cells grown on permeable solid support (e.g. on porous membranes). The vectorial transport assay described so far [Evers, R. et al, J. Clin. Invest. 97, 1211–1218 (1996)], though being suitable to experimentally investigate the localization and activity of transport proteins like MRP1 and MRP2, suffers from the serious drawback of using exclusively radioactively labeled substrates for detection which, of course, requires the application of extra laboratory safety equipment and regulations. Another drawback is the previously described vectorial transport assay is that certain labeled substrates used for the assessment of the activity of the intracellular transport proteins of interest might be able to pass the tight junctions contacting the cells of the confluent culture used and, therefore, can get to the opposite compartment even without being transported, thus making very complicated to develop a suitable quantitative assay for the exact determination of the activity of the transport proteins of interest.
It is, therefore, an object of the present invention is to provide simple qualitative and quantitative vectorial transport assay methods that are significantly easier to perform and automate than the previously described ones and can also be made quantitative by only detecting the transcellular export (and not the transport going possibly through the tight junctions of the polarized culture and thereby avoiding uptake and excretion by the cells). Another object of the present invention is to provide assay methods for the identification of inhibitors and activators of transport proteins of interest and, also, assay methods for the fast and easy assessment of the localization of transport proteins of interest in the basolateral and/or apical plasma membrane.
Calcein is a well known fluorescent substance (commercialized by Molecular Probes, Inc. USA) and the non-fluorescent acetoxy-methyl ester derivative of said compound (calcein AM) is known to be cell permeable due to its hydrophobic character. We have previously developed different assay arrangements using calcein AM for the assessment of the activity of MDR1 or other transport proteins which can extrude calcein AM from the cells but can not extrude calcein itself (U.S. Pat. No. 5,872,014; European Patent No. 0,784,699). We now have recognized that calcein and calcein AM (and other compounds of similar behavior) can also be used to elaborate assay methods for the assessment of the activity of MRP1, MRP2 and similar transport proteins which can effectively extrude calcein out from the cells.